1. Field of the Invention
This invention relates to novel procainamide and N-acetylprocainamide (NAPA) derivatives pertaining to immunoassays for determining such drugs in liquid media such as biological fluids. The derivatives include immunogens used to stimulate production of antibodies to the drugs in host animals by conventional techniques. Also provided are labeled conjugates used as reagents, along with the antibodies, in particularly preferred immunoassays. Intermediates in the synthesis of the aforementioned immunogens and labeled conjugates are also provided.
The cardiac depressant drugs procainamide and N-acetylprocainamide are used clinically to treat or prevent cardiac arrhythmia (J. Kosh-Weser, et al, New England J. Med. 281: 1253 (1969); J. Koch-Weser and S. W. Klein, J. Am. Med. Assoc. 215: 1454 (1971)]. N-acetylprocainamide (abbreviated as NAPA and also known as acecainide) is the major metabolite of procainamide in man. The concentration of this metabolite in the plasma of patients receiving procainamide often exceeds the concentration of the parent drug itself. Metabolism is by in vivo acetylization and much genetic-based variation has been observed in the rate at which individual patients transform the drug to its metabolite [D. Drayer, et al, Drug Metab. Rev. 10: 239 (1979)]. This phenomenon is of importance in the clinical use of the drugs because of the lower incidence of side effects associated with NAPA. Both the therapeutic usefulness and the toxicity of the drugs are better correlated with their blood levels than with their dosages. The relationship between the amount of drug administered and the blood levels is quite variable. It is influenced by completeness of absorption, distribution characteristics and rates of metabolism and excretion.
2. Description of the Prior Art
Because of these considerations, numerous analytical methods have been developed to determine the blood levels of these drugs, including high pressure liquid chromatography (HPLC) [L. R. Shukus, et al, Clin. Chem. 23: 705 (1977)], homogeneous enzyme immunoassay [C. B. Walberg, "Proc. Intl. Sym. Enzyme Labeled Hormones and Drugs", S. B. Pal, ed., W. deGruyter & Co. (Berlin, 1978), p. 429], and quantitative thin layer chromatography (TLC) [B. Wesley-Hadzya and A. M. Mattocks, J. Chromat. 143: 307 (1977)].
The preparation of antibodies to procainamide and NAPA for use in immunoassays to determine the drugs has been accomplished in the prior art by essentially two different approaches. One approach has been to couple procainamide through the ring amino group by diazotization subsequent condensation to an albumin carrier [A. S. Russel, et al, Clin. Exp. Immunol. 3: 901 (1968) and Mojaverian et al, J. Pharm. Sci. 69: 721 (1980)]. The resulting antibodies show a high degree of cross-reactivity with NAPA and would therefore be unsuited for use in immunoassays specific for one or the other drug.
The second approach involves coupling of the drugs at the complete opposite end of their structures, at the N-diethylamino group, by modification of one of the ethyl substituents for subsequent coupling to a carrier [U.S. Pat. No. 4,235,969]. As a result, antibodies are raised against an immunogen in which a major functional group of the drugs has been modified in order to couple them to the carrier.
The state-of-the-art of preparing antibodies to haptens such as drugs is represented by Weinryb et al, Drug Metabolism Reviews 10: 271 (1979); Playfair et al, Br. Med. Bull. 30: 24 (1974); Broughton et al, Clin. Chem. 22: 726 (1976); and Butler, J. Immunol. Meth. 7: 1 (1975) and Pharmacol Rev. 29(2): 103 (1978).
Labeled conjugates, comprising the analyte or a derivative or other analog thereof, coupled to a labeling substance are variously described in the literature, e.g., U.S. Pat. Nos. 4,279,992; 4,182,856; 4,259,233; and 4,292,425 wherein the label is the fluorogenic enzyme substrate .beta.-galactosyl-umbelliferone.